In this competing renewal application we request support for our studies on late events in retrovirus assembly. In the previous funding cycle, we identified and characterized several cellular proteins that are recruited to sites of retrovirus assembly by viral late budding (L)-domains. These proteins are either (i) components of the class E vacuolar protein sorting (VPS) pathway that is normally involved in the manipulation of endosomes and multivesicular bodies (MVB) or (ii) ubiquitin ligases. For several of these factors we were able to demonstrate a critical role in the release of infectious retroviral particles. Several mechanistic questions remain, however, and recently we have identified additional cellular factors that seem very likely to be involved in the late stages of the construction and release of retrovirus particles, and/or endosome/MVB biogenesis. In the next grant period, we will attempt to elucidate how previously defined and newly identified cellular factors participate in the late stages of retrovirus assembly and related cellular functions by pursuing two specific aims. Specific Aim 1 is to understand how specific endosomal proteins are incorporated into HIV-1 particles and their role in generating infectious virions. Specific aim 2 is to determine the role of ubiquitin, ubiquitin ligases and novel ubiquitin ligase binding proteins, discovered during the previous funding period, in retroviral budding and trafficking of endocytosed proteins. Execution of these two aims should result in significant progress toward our long term goal of understanding how cellular and viral factors cooperate in the construction of complete, infectious retroviral particles. Understanding these events could potentially provide new opportunities to interfere in these processes for the treatment of diseases caused by HIV and other enveloped viruses. Public health relevance: Many enveloped viruses, for example HIV-1, are responsible for life threatening infections. To spread from cell to cell they make use of cellular proteins to facilitate the release of accurately assembled virus particles. Understanding how this system works could give opportunities to intervene with targeted therapeutics.